Natural human interferon β (IFN-β) is a glycoprotein consisting of 166 amino acid residues. Interferon β belongs to the cytokine family, and is known to be involved in immunomodulation action, antiviral activity, and cell proliferation suppression action. Moreover, human interferon β has three Cys at positions 17, 31, and 141 of its amino acid sequence, and has a monoantennary complex-type N-linked oligosaccharide at asparagine at position 80. Moreover, it is known to have a disulfide bond at Cys at positions 31 and 141. Interferon β as a pharmaceutical is manufactured utilizing a cell expression system, and is classified into IFN-β-1a or IFN-β-1b depending on the difference in the host for expression. While IFN-β-1a is a glycoprotein, IFN-β-1b does not have an oligosaccharide. Further, IFN-β-1a having a sugar chain is known to have a more potent effect in immunogenicity, antiviral activity, and antitumor property compared to IFN-β-1b.
It is known that the sugar chain structure comprised in the glycoprotein has a strong influence on pharmacokinetics. In particular, the presence or absence of a sialic acid present at the non-reducing terminal of the sugar chain is known to have an influence on the extension of half-life in blood. However, previously biosynthesized IFN-β-1a is reported to have ununiform sugar chain structure in the polypeptide (for example, the result of analyzing the sugar chain structure ununiformity by CE-TOF-MS is described in Non-Patent Literature 1). Moreover, isolation of IFN—β having substantially uniform sugar chain structure from a synthesized IFN—β or a naturally-occurring IFN—β has not been reported. This has thus been an obstacle in presuming the sugar chain structure important for bioactivity.
In recent years, the biotechnology of utilizing a cell expression system has enabled the manufacture of bioactive protein formulations including interferons, such as insulin, erythropoietin, and G-CSF. In these protein formulations, it is reported that the glycosylation pattern of the protein renders variety to physical or chemical properties of the protein such as the folding step, or conformation property, stability, immune reaction, half-life in blood, and function of the protein in the biological system (Non-Patent Literature 2). Moreover, considering the crucial immunogenicity caused by a non-human-type sugar chain, the structure of the sugar chain added to these protein formulations is preferably a human-type sugar chain.
Such glycoprotein formulations have been manufactured by the cell expression system as the only method. However, as described above, the technology of such a cell expression system could not go the length of controlling the sugar chain structure, thereby causing ununiformity in the sugar chain structure in the glycoprotein manufactured (e.g. Non-Patent Literature 3). For this reason, there were problems such as variability of the quality between production lots or the inability to optimize the sugar chain. Accordingly, a method for preparing a uniform glycoprotein wherein the sugar chain structure is easily adjustable has been long desired, but currently, there is no report of synthesizing a human-type uniform glycoprotein showing bioactivity in vivo by chemical synthesis.
Moreover, a bioactive glycoprotein manufactured by a technology by such a cell expression system may comprise a virus or genetic material. Moreover, there is a possibility of contamination with such genetic material etc. also in the case of synthesizing a bioactive glycoprotein using a sugar chain prepared from a biologically-derived sample. Application of heat treatment that can disintegrate these genetic materials is desired in order to manufacture a safe protein formulation. However, currently, heat treatment on a bioactive glycoprotein formulation will inactivate the glycoprotein, and application of heat treatment has not been reported.